CN118284625A - Combination therapy of anti-TYRP 1/anti-CD 3 bispecific antibodies and TYRP 1-specific antibodies - Google Patents

Abstract

The present invention relates to combination therapy of a bispecific antibody that binds to human TYRP1 and CD3 with a second TYRP1-specific antibody.

Description

Combination therapy of anti-TYRP1/anti-CD3 bispecific antibody and TYRP1-specific antibody

Technical Field

The present invention relates to a combination therapy of a bispecific antibody binding to human TYRP1 and CD3 and a TYRP1-specific antibody.

Background technique

Cancer is the leading cause of death in economically developed countries and the second leading cause of death in developing countries. Despite recent advances in chemotherapy and the development of drugs that target and interfere with growth signal transduction and regulation in cancer cells at the molecular level, the prognosis for patients with advanced cancer remains generally poor. Therefore, there is an urgent medical need to continuously develop new therapies that can be added to existing treatments to prolong survival without causing unacceptable toxicity.

CD3 (cluster of differentiation 3) is a protein complex composed of four subunits, namely CD3γ chain, CD3δ chain and two CD3ε chains. CD3 binds to the T cell receptor and ζ chain to generate activation signals in T lymphocytes. CD3 has been widely explored as a drug target. Monoclonal antibodies targeting CD3 have been used as immunosuppressant therapy for autoimmune diseases (such as type 1 diabetes) or in the treatment of transplant rejection. In 1985, the CD3 antibody muromonab-CD3 (OKT3) became the first monoclonal antibody approved for clinical use in humans.

Currently, the latest application of CD3 antibodies is in the form of bispecific antibodies, which bind to CD3 on one side and to tumor cell antigens on the other side (Clynes and Desjarlais (2019) Annu. Rev. Med. 70: 437-50). The simultaneous binding of this antibody to its two targets will force a temporary interaction between the target cell and the T cell, thereby activating any cytotoxic T cells and subsequently lysing the target cell. To this end, a bispecific antibody that binds CD3 and the tumor cell antigen TYRP1 has been developed; as described, for example, in WO 2020/127619 A1. TYRP1 is present as a target in melanoma cells and melanocytes, participates in melanin synthesis, and also affects the proliferation and survival of human melanocytes. TYRP1 antibodies have been previously described (Boross et al. (2014) Immunol Lett. 160 (2): 151-7) and tested in clinical trials (Khalil et al. (2016) Clin Cancer Res. 22 (21): 5204-5210). For use in mouse models, TYRP1 and T cell bispecific surrogate antibodies have been described (Benonisson et al. (2019) Mol Cancer Ther. (2): 312-322; Labrijn et al. (2017) Sci Rep. 7(1): 2476), where they mediated anti-tumor efficacy but failed to induce long-term responses/cures.

There remains a need for new compounds and combinations that could significantly advance patient treatment. Accordingly, we describe herein novel combination therapies of TYRP1 antibodies and bispecific antibodies that bind to TYRP1 and CD3.

Summary of the invention

The present invention comprises a combination of an anti-TYRP1/anti-CD3 bispecific antibody for use as a combination therapy for treating cancer, or for use as a combination therapy for preventing or treating metastasis, and a second TYRP1-specific antibody, wherein the anti-TYRP1/anti-CD3 bispecific antibody comprises a first antigen-binding portion that specifically binds to TYRP1 and a second antigen-binding portion that specifically binds to CD3, the first antigen-binding portion comprising a heavy chain variable domain VH of SEQ ID NO: 1 and a light chain variable domain VL of SEQ ID NO: 2, the second antigen-binding portion comprising a heavy chain variable domain VH of SEQ ID NO: 3 and a light chain variable domain VL of SEQ ID NO: 4, and wherein the second TYRP1-specific antibody comprises an antigen-binding portion that specifically binds to TYRP1.

In addition, the present invention provides use of a combination of an anti-TYRP1/anti-CD3 bispecific antibody and a second TYRP1-specific antibody in the manufacture of a medicament for treating cancer, wherein the anti-TYRP1/anti-CD3 bispecific antibody comprises a first antigen-binding portion that specifically binds to TYRP1 and a second antigen-binding portion that specifically binds to CD3, the first antigen-binding portion comprises a heavy chain variable domain VH of SEQ ID NO: 1 and a light chain variable domain VL of SEQ ID NO: 2, the second antigen-binding portion comprises a heavy chain variable domain VH of SEQ ID NO: 3 and a light chain variable domain VL of SEQ ID NO: 4, and wherein the second TYRP1-specific antibody comprises an antigen-binding portion that specifically binds to TYRP1.

In addition, the present invention provides a method for treating cancer in an individual, the method comprising administering to the individual a therapeutically effective amount of an anti-TYRP1/anti-CD3 bispecific antibody in combination with a second TYRP1-specific antibody, wherein the anti-TYRP1/anti-CD3 bispecific antibody comprises a first antigen-binding portion that specifically binds to TYRP1 and a second antigen-binding portion that specifically binds to CD3, the first antigen-binding portion comprising a heavy chain variable domain VH of SEQ ID NO: 1 and a light chain variable domain VL of SEQ ID NO: 2, the second antigen-binding portion comprising a heavy chain variable domain VH of SEQ ID NO: 3 and a light chain variable domain VL of SEQ ID NO: 4, and wherein the second TYRP1-specific antibody comprises an antigen-binding portion that specifically binds to TYRP1.

In one aspect, the second TYRP1-specific antibody comprises a heavy chain variable domain VH of SEQ ID NO:1 and a light chain variable domain VL of SEQ ID NO:2.

In a further aspect, the anti-TYRP1/anti-CD3 bispecific antibody is of human IgG ₁ or human IgG ₄ subclass. In one aspect, the second TYRP1-specific antibody is of human IgG ₁ subclass.

In one aspect, the anti-TYRP1/anti-CD3 bispecific antibody has reduced effector function or minimal effector function. In another aspect, minimal effector function is caused by an effector-free Fc mutation. In one aspect, the effector-free Fc mutation is L234A/L235A or L234A/L235A/P329G or N297A or D265A/N297A.

In one aspect, the second TYRP1 -specific antibody comprises an Fc domain with improved effector function, in particular improved ADCC function. In one aspect, the second TYRP1 -specific antibody is afucosylated.

In one aspect, the present invention provides an anti-TYRP1/anti-CD3 bispecific antibody for use in combination with a second TYRP1-specific antibody according to any one of the preceding aspects, a use or a method, wherein the anti-TYRP1/anti-CD3 bispecific antibody comprises i) a polypeptide sequence of SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8, ii) a polypeptide sequence of SEQ ID NO: 5 and SEQ ID NO: 6 and SEQ ID NO: 7 and SEQ ID NO: 8, or iii) a polypeptide sequence of SEQ ID NO: 9 and SEQ ID NO: 10 and SEQ ID NO: 11 and SEQ ID NO: 12.

In another aspect, the present invention provides an anti-TYRP1/anti-CD3 bispecific antibody for use, in combination, use or method with a second TYRP1-specific antibody according to any one of the preceding aspects, wherein the anti-TYRP1/anti-CD3 bispecific antibody comprises i) a polypeptide sequence of SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8, ii) a polypeptide sequence of SEQ ID NO: 5 and SEQ ID NO: 6 and SEQ ID NO: 7 and SEQ ID NO: 8, or iii) a polypeptide sequence of SEQ ID NO: 9 and SEQ ID NO: 10 and SEQ ID NO: 11 and SEQ ID NO: 12, and wherein the second TYRP1-specific antibody comprises i) a polypeptide sequence of SEQ ID NO: 13 or SEQ ID NO: 14, or ii) a polypeptide sequence of SEQ ID NO: 13 and SEQ ID NO: 14; or iii) a polypeptide sequence of SEQ ID NO: 15 and SEQ ID NO: 16.

In another aspect, the present invention provides a combination of an anti-TYRP1/anti-CD3 bispecific antibody and a second TYRP1-specific antibody for use in: i) inhibiting tumor growth in a tumor; and/or ii) increasing the median and/or overall survival of subjects having a tumor; wherein the anti-TYRP1/anti-CD3 bispecific antibody comprises i) a polypeptide sequence of SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8, ii) a polypeptide sequence of SEQ ID NO: 5 and SEQ ID NO: 6 and SEQ ID NO: 7 and SEQ ID NO: 8, or iii) a polypeptide sequence of SEQ ID NO: 9 and SEQ ID NO: 10 and SEQ ID NO: 11 and SEQ ID NO: 12, and wherein the second TYRP1-specific antibody comprises i) a polypeptide sequence of SEQ ID NO: 13 or SEQ ID NO: 14, or ii) a polypeptide sequence of SEQ ID NO: 13 and SEQ ID NO: 14; or iii) a polypeptide sequence of SEQ ID NO: 15 and SEQ ID NO: 16.

In a further aspect, the present invention provides an anti-TYRP1/anti-CD3 bispecific antibody, use or method in combination with a second TYRP1-specific antibody according to any of the aforementioned aspects, wherein the cancer is selected from the group consisting of breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma cancer, bladder cancer, kidney cancer, renal cancer, liver cancer, head and neck cancer, colorectal cancer, melanoma, pancreatic cancer, gastric cancer, esophageal cancer, mesothelioma, prostate cancer, leukemia, lymphoma, myeloma. In one aspect, the patient is treated or pretreated with immunotherapy. In one aspect, the immunotherapy comprises adoptive cell transfer, administration of monoclonal antibodies, administration of cytokines, administration of cancer vaccines, T cell engagement therapy, or any combination thereof. In one aspect, adoptive cell transfer comprises administration of T cells (CAR T cells) expressing chimeric antigen receptors, T cell receptor (TCR) modified T cells, tumor infiltrating lymphocytes (TILs), natural killer cells modified by chimeric antigen receptors (CARs), cells transduced by T cell receptors (TCRs), or dendritic cells, or any combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Shows the results of efficacy experiments evaluating muTYRP1-IgG and muTYRP1-TCB as single agents and combination drugs. The B16-muFAP-Fluc double-transfected melanoma cell line was intravenously injected into Black 6 albino mice to study survival in a syngeneic model of lung metastasis. The amount of antibody (mg/kg) injected per mouse is as follows: 20mg/kg muTYRP1-IgG and 10mg/kg muTYRP1-TCB. The antibodies were injected intravenously once a week for 4 weeks. Significantly superior median and overall survival were observed in the 10mg/kg muTYRP1-TCB + 20mg/kg muTYRP1-IgG combination group compared to the single drug and vehicle groups tested.

Detailed ways

Anti-TYRP1/anti-CD3 bispecific antibodies are described in WO 2020/127619 A1.

The anti-TYRP1/anti-CD3 bispecific antibodies used in the combination therapy described herein comprise a first antigen binding portion capable of binding to TYRP1 and a second antigen binding portion capable of binding to CD3. The anti-TYRP1/anti-CD3 bispecific antibodies used in the combination therapies described herein may comprise a first antigen binding portion comprising a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 1, or a variant thereof that retains functionality, and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 2, or a variant thereof that retains functionality, and a second antigen binding portion comprising a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 3, or a variant thereof that retains functionality, and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the sequence of SEQ ID NO: 4. NO:4 sequence or a variant thereof that retains functionality having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to a light chain variable region sequence. The anti-TYRP1/anti-CD3 bispecific antibody used in the combination therapy described herein may comprise a first antigen binding portion comprising a heavy chain variable domain VH of SEQ ID NO:1 and a light chain variable domain VL of SEQ ID NO:2, and a second antigen binding portion comprising a heavy chain variable domain VH of SEQ ID NO:3 and a light chain variable domain VL of SEQ ID NO:4.

The anti-TYRP1/anti-CD3 bispecific antibodies used in the combination therapy described herein may include a third antigen binding moiety that is identical to the first antigen binding moiety. In one embodiment, the first antigen binding moiety and the third antigen binding moiety that bind to TYRP1 are conventional Fab molecules. In such an embodiment, the second antigen binding moiety that binds to CD3 is a crossover Fab molecule, i.e., a Fab molecule in which the variable domains VH and VL of the Fab heavy chain and light chain are exchanged/replaced with each other.

The anti-TYRP1/anti-CD3 bispecific antibodies for use in combination therapy may comprise amino acid substitutions in the Fab molecules contained therein that are particularly effective in reducing mispairing of the light chain with an unmatched heavy chain (Bence-Jones type byproducts), which mispairing may occur in the production of Fab-based multispecific antibodies, the bi/multispecific antigen-binding molecule having a VH/VL exchange in one (or multiple, if the molecule comprises more than two antigen-binding Fab molecules) of its binding arms (see also PCT Publication No. WO 2015/150447, in particular the examples therein, the entire contents of which are incorporated herein by reference). The ratio of the desired (multispecific) antibody to undesirable byproducts, in particular Bence Jones type byproducts occurring in multispecific antibodies having a VH/VL domain exchange in one of the binding arms of the (multispecific) antibody, can be improved by introducing charged amino acids with opposite charges at specific amino acid positions in the CH1 and CL domains (sometimes referred to herein as "charge modification").

Thus, an anti-TYRP1/anti-CD3 bispecific antibody for use in combination therapy, wherein the first and second antigen-binding moieties (and the third antigen-binding moiety when present) of the (multispecific) antibody are Fab molecules, and in one of the antigen-binding moieties (particularly the second antigen-binding moiety) the variable domains VL and VH of the Fab light chain and the Fab heavy chain are replaced with each other, in the constant domain CL of the first antigen-binding moiety (and the third antigen-binding moiety when present), the amino acid at position 124 and the amino acid at position 213 can be substituted with a positively charged amino acid (according to Kabat numbering), and wherein in the constant domain CH1 of the first antigen-binding moiety (and the third antigen-binding moiety when present), the amino acid at position 147 and the amino acid at position 213 can be substituted with a negatively charged amino acid (according to the Kabat EU index numbering). The constant domains CL and CH1 of the antigen-binding moiety with VH/VL exchange are not replaced with each other (i.e. remain unexchanged). The amino acid at position 124 and the amino acid at position 213 of the constant domain CL may be independently substituted with lysine (K), arginine (R) or histidine (H) (according to Kabat numbering), and the amino acid at position 147 and the amino acid at position 213 of the constant domain CH1 may be independently substituted with glutamic acid (E) or aspartic acid (D) (according to Kabat EU index numbering). The anti-TYRP1/anti-CD3 bispecific antibody for use in combination therapy may have, in the constant domain CL of the first antigen-binding portion (and the third antigen-binding portion when present), the amino acid at position 124 substituted with lysine (K) (according to Kabat numbering) and the amino acid at position 123 substituted with arginine (R) (according to Kabat numbering), and in the constant domain CH1 of the first antigen-binding domain (and the third antigen-binding domain when present), the amino acid at position 147 substituted with glutamic acid (E) (according to Kabat EU index numbering) and the amino acid at position 213 substituted with glutamic acid (E) (according to Kabat EU index numbering).

The anti-TYRP1/anti-CD3 bispecific antibodies used in the combination therapy described herein may have the sequences shown in SEQ ID NOs: 5, 6, 7 and 8, or variants thereof that retain functionality.

The anti-TYRP1/anti-CD3 bispecific antibodies having the sequences shown in SEQ ID NOs: 5, 6, 7 and 8 are referred to herein as "TYRP1 TCB" or "TYRP1-TCB". The anti-TYRP1/anti-CD3 bispecific antibodies having the sequences shown in SEQ ID NOs: 9, 10, 11 and 12 are referred to herein as "muTYRP1 TCB", which is a mouse surrogate.

Anti-TYRP1/anti-CD3 bispecific antibodies may include an Fc domain that includes modifications that promote heterodimerization of two different polypeptide chains. "Modifications that promote heterodimerization" are manipulations of the peptide backbone or post-translational modifications of a polypeptide that reduce or prevent the polypeptide from associating with the same polypeptide to form a homodimer. As used herein, modifications that promote heterodimerization particularly include separate modifications to each of the two polypeptides required to form a dimer, wherein the modifications are complementary to each other to promote the association of the two polypeptides. For example, modifications that promote heterodimerization can change the structure or charge of one or both of the polypeptides required to form a dimer so as to make their association spatially or electrostatically favorable, respectively. Heterodimerization occurs between two different polypeptides, such as two subunits of an Fc domain, wherein the subunits are not identical. In the bispecific antibodies according to the present invention, modifications that promote heterodimerization are located in the Fc domain. In some embodiments, modifications that promote heterodimerization include amino acid mutations, particularly amino acid substitutions. In specific embodiments, modifications that promote heterodimerization include separate amino acid mutations, particularly amino acid substitutions, to each of the two subunits of the Fc domain. The most extensive protein-protein interaction site between the two polypeptide chains of the human IgG Fc domain is in the CH3 domain of the Fc domain. Therefore, in one embodiment, the modification is located in the CH3 domain of the Fc domain. In a specific embodiment, the modification is a knob-hole modification, which includes a knob modification in one of the two subunits of the Fc domain and a hole modification in the other of the two subunits of the Fc domain.

The knob-and-hole technique is described in, for example, US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001). Typically, the method involves introducing a protrusion ("knob") at the interface of a first polypeptide and introducing a corresponding cavity ("hole") in the interface of a second polypeptide, such that the protrusion can be positioned in the cavity to promote the formation of heterodimers and hinder the formation of homodimers. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). A compensatory cavity having the same or similar size as the protrusion is created in the interface of the second polypeptide by replacing a large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). The protrusion and cavity can be prepared by altering the nucleic acid encoding the polypeptide, for example, by site-specific mutagenesis or by peptide synthesis. In a specific embodiment, the knob modification comprises the amino acid substitution T366W in one of the two subunits of the Fc domain, and the hole modification comprises the amino acid substitutions T366S, L368A and Y407V in the other of the two subunits of the Fc domain. In another specific embodiment, the subunit comprising the knob modified Fc domain additionally comprises the amino acid substitution S354C, and the subunit comprising the hole modified Fc domain additionally comprises the amino acid substitution Y349C. The introduction of these two cysteine residues results in the formation of a disulfide bridge between the two subunits of the Fc region, thereby further stabilizing the dimer (Carter, J Immunol Methods 248, 7-15 (2001)). The numbering of amino acid residues in the Fc region is according to the EU numbering system, which is also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. As used herein, a "subunit" of an Fc domain refers to one of the two polypeptides that form a dimeric Fc domain, i.e., a polypeptide comprising the C-terminal constant region of an immunoglobulin heavy chain, which polypeptide is capable of stable self-association. For example, a subunit of an IgG Fc domain comprises the IgG CH2 and IgG CH3 constant domains.

In alternative embodiments, modifications that promote heterodimerization of two non-identical polypeptide chains include modifications that mediate electrostatic steering effects, such as described in WO 2009/089004. Typically, the method involves replacing one or more amino acid residues at the interface of the two polypeptide chains with charged amino acid residues such that homodimer formation becomes electrostatically unfavorable, but heterodimerization is electrostatically favorable.

Compared to non-engineered Fc domains, the Fc domain of the anti-TYRP1/anti-CD3 bispecific antibody can be engineered to have an altered binding affinity to Fc receptors, in particular an altered binding affinity to Fcγ receptors, as described in WO 2012/146628. The binding of the Fc domain to complement components, in particular C1q, can be altered, as described in WO 2012/146628. The Fc domain confers favorable pharmacokinetic properties to the antibody, including a long serum half-life and a favorable tissue-blood distribution ratio that contribute to good accumulation in target tissues. However, at the same time, it may result in undesirable targeting to cells expressing Fc receptors rather than preferred cells with antigens.

Therefore, the Fc domain of the anti-TYRP1/anti-CD3 bispecific antibody can be engineered to have a reduced binding affinity to the Fc receptor. In one such embodiment, the Fc domain comprises one or more amino acid mutations that reduce the binding affinity of the Fc domain to the Fc receptor. Typically, the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. In one embodiment, the amino acid mutation reduces the binding affinity of the Fc domain to the Fc receptor by at least 2 times, at least 5 times, or at least 10 times. In the embodiment where there is more than one amino acid mutation that reduces the binding affinity of the Fc domain to the Fc receptor, the combination of these amino acid mutations can reduce the binding affinity of the Fc domain to the Fc receptor by at least 10 times, at least 20 times, or even at least 50 times. In one embodiment, compared to an antibody comprising a non-engineered Fc domain, an antibody comprising an engineered Fc domain exhibits less than 20%, particularly less than 10%, and more particularly less than 5% of the binding affinity to the Fc receptor. In one embodiment, the Fc receptor is an activated Fc receptor. In a specific embodiment, the Fc receptor is an Fcγ receptor, more specifically an FcγRIIIa, FcγRI or FcγRIIa receptor. Preferably, the binding to each of these receptors is reduced. In some embodiments, the binding affinity to complement components, particularly the binding affinity to C1q, is also reduced. In one embodiment, the binding affinity to the neonatal Fc receptor (FcRn) is not reduced. When the Fc domain (or an antibody comprising the Fc domain) shows a binding affinity of greater than about 70% of the Fc domain (or an antibody comprising the Fc domain) of a non-engineered form to FcRn, a substantially similar binding to FcRn is achieved, i.e., the binding affinity of the Fc domain to the receptor is maintained. The Fc domain or an antibody of the present invention comprising the Fc domain may show such an affinity greater than about 80%, and even greater than about 90%. In one embodiment, the amino acid mutation is an amino acid substitution. In one embodiment, the Fc domain comprises an amino acid substitution at position P329. In a more specific embodiment, amino acid substitution is P329A or P329G, particularly P329G. In one embodiment, the Fc domain comprises further amino acid substitution at a position selected from S228, E233, L234, L235, N297 and P331. In a more specific embodiment, further amino acid substitution is S228P, E233P, L234A, L235A, L235E, N297A, N297D or P331S. In a specific embodiment, the Fc domain comprises amino acid substitution at position P329, L234 and L235. In a more specific embodiment, the Fc domain comprises amino acid mutations L234A, L235A and P329G (LALA P329G). This combination of amino acid substitutions almost completely eliminates the Fc gamma receptor binding of human IgG Fc domains, as described in WO 2012/130831, which is incorporated herein by reference in its entirety. WO 2012/130831 also describes methods for preparing such mutant Fc domains and methods for determining their properties (such as Fc receptor binding or effector function). The numbering of amino acid residues in the Fc region is according to the EU numbering system, which is also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

Mutant Fc domains can be prepared by amino acid deletion, substitution, insertion or modification using genetic or chemical methods well known in the art and as described in WO 2012/146628. Genetic methods may include site-specific mutagenesis of the encoding DNA sequence, PCR, gene synthesis, etc. The correct nucleotide changes can be verified, for example, by sequencing.

In one embodiment, the Fc domain is engineered to have reduced effector function compared to a non-engineered Fc domain, as described in WO 2012/146628. Reduced effector function may include, but is not limited to, one or more of reduced complement dependent cytotoxicity (CDC), reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen presenting cell uptake of antigen, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling-induced apoptosis, reduced cross-linking of target-bound antibodies, reduced dendritic cell maturation, or reduced T cell sensitization.

Compared to IgG ₁ antibodies, IgG ₄ antibodies exhibit reduced binding affinity to Fc receptors and reduced effector functions. Therefore, in some embodiments, the Fc domain of the antibody of the present invention is an IgG ₄ Fc domain, particularly a human IgG ₄ Fc domain. In one embodiment, the IgG ₄ Fc domain comprises an amino acid substitution at position S228, particularly an amino acid substitution S228P. In order to further reduce its binding affinity to Fc receptors and/or its effector functions, in one embodiment, the IgG ₄ Fc domain comprises an amino acid substitution at position L235, particularly an amino acid substitution L235E. In another embodiment, the IgG _{4 Fc domain comprises an amino acid substitution at position P329, particularly an amino acid substitution P329G. In a specific embodiment, the IgG 4 Fc} domain comprises an amino acid substitution at positions S228, L235 and P329, particularly an amino acid substitution S228P, L235E and P329G. Such IgG ₄ Fc domain mutants and their Fcγ receptor binding properties are described in European Patent Application No. WO 2012/130831, the entire contents of which are incorporated herein by reference.

The TYRP1-specific antibodies used in the combination therapy described herein include an antigen binding portion capable of binding to TYRP1. The TYRP1-specific antibodies used in the combination therapy described herein may include an antigen binding portion comprising a heavy chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the SEQ ID NO: 1 sequence or a variant thereof that retains functionality, and a light chain variable region sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the SEQ ID NO: 2 sequence or a variant thereof that retains functionality. The TYRP1-specific antibodies used in the combination therapy described herein may include an antigen binding portion that includes a heavy chain variable domain VH of SEQ ID NO: 1 and a light chain variable domain VL of SEQ ID NO: 2.

TYRP1-specific antibodies having sequences as shown in SEQ ID NO: 13 and SEQ ID NO: 14 are referred to herein as "TYRP1 IgG" or "TYRP1-IgG". TYRP1-specific antibodies having sequences as shown in SEQ ID NO: 15 and SEQ ID NO: 16 are referred to herein as "muTYRP1 IgG" or "muTYRP1-IgG", which are murine alternatives.

The TYRP1-specific antibody used in the combination therapy described herein may be an antibody of the IgG ₁ subclass. The Fc domain of the TYRP1-specific antibody may have ADCC activity in the presence of human effector cells, or have increased ADCC activity in the presence of human effector cells, compared to an otherwise identical antibody comprising a human wild-type IgG ₁ Fc region.

In certain embodiments, the antibodies provided herein are altered to increase or decrease the extent of antibody glycosylation. Glycosylation sites can be conveniently added or deleted to an antibody by altering the amino acid sequence to create or remove one or more glycosylation sites.

When antibody comprises Fc district, the oligosaccharide connected thereto can be changed.Native antibodies produced by mammalian cells usually comprise the biantennary oligosaccharide of side chain, and this biantennary oligosaccharide is usually connected to the Asn297 of the CH2 domain of Fc district by N-bonding.See, for example, Wright et al. TIBTECH 15:26-32 (1997).Oligosaccharide can include various carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and the fucose of GlcNAc in the " trunk " of biantennary oligosaccharide structure.In certain embodiments, the oligosaccharide in the antibody of the present invention can be modified, so as to produce antibody variants with some improved characteristics.

In one embodiment, antibody variants are provided having defucosylated oligosaccharides, i.e., oligosaccharide structures lacking fucose attached (directly or indirectly) to the Fc region. Such defucosylated oligosaccharides (also referred to as "defucosylated" oligosaccharides) are particularly N-linked oligosaccharides that lack a fucose residue attached to the first GlcNAc in the stem of the biantennary oligosaccharide structure. In one embodiment, antibody variants are provided that have an increased proportion of defucosylated oligosaccharides in the Fc region compared to a native or parent antibody. For example, the proportion of defucosylated oligosaccharides can be at least about 20%, at least about 40%, at least about 60%, at least about 80%, or even about 100% (i.e., the absence of fucosylated oligosaccharides). The percentage of defucosylated oligosaccharides, as described, for example, in WO 2006/082515, as measured by MALDI-TOF mass spectrometry, is the (average) amount of oligosaccharides lacking a fucose residue relative to the sum of all oligosaccharides (e.g., complex, hybrid and high mannose structures) linked to Asn 297. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (EU numbering of residues in the Fc region); however, due to minor sequence variations in antibodies, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such antibodies having an increased proportion of defucosylated oligosaccharides in the Fc region may have improved FcγRIIIa receptor binding and/or improved effector function, in particular improved ADCC function. See, for example, US2003/0157108 and US2004/0093621.

Examples of cell lines capable of producing antibodies with reduced fucosylation include Lec13 CHO cells lacking protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); US 2003/0157108; and WO 2004/056312, particularly in Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87:614-622 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2004/056312, particularly in Example 11). 2003/085107), or cells with reduced or abolished GDP-fucose synthesis or transporter activity (see, e.g., US2004259150, US2005031613, US2004132140, US2004110282).

In another embodiment, the antibody variant provides a bisected oligosaccharide, for example, wherein the biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. As described above, such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described in, for example, Umana et al., Nat Biotechnol 17, 176-180 (1999); Ferrara et al., Biotechn Bioeng 93, 851-861 (2006); WO 99/54342; WO 2004/065540, WO 2003/011878.

Also provided are antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region. Such antibody variants may have improved CDC function. Such antibody variants are described in, for example, WO 1997/30087, WO 1998/58964, and WO 1999/22764.

The Fc domain of the TYRP1-specific antibody may comprise one or more amino acid substitutions that improve ADCC by increasing binding to an Fc receptor, preferably to FcγRIII. The Fc domain may comprise amino acid substitutions at positions 298, 333, and/or 334 of the Fc region (EU numbering of residues) (Shields et al., J Biol Chem. 276(9): 6591-604(2001)). Further examples of such Fc variants are described in Wang et al., Protein Cell. 9(1): 63-73(2018); and Nordstrom et al., Breast Cancer Res. 13(6): R123(2011).

In certain embodiments, the TYRP1-specific antibody variants comprising the Fc region described herein are capable of binding to FcγRIII. In certain embodiments, the antibody variants comprising the Fc region described herein have ADCC activity in the presence of human effector cells, or have increased ADCC activity in the presence of human effector cells, compared to an otherwise identical antibody comprising a human wild-type IgG ₁ Fc region.

definition

The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and antibody fragments, as long as they exhibit the desired antigen-binding activity. "Antibody fragment" refers to a molecule other than an intact antibody, which comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab') ₂ , diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and single-domain antibodies. For a review of certain antibody fragments, see Hudson et al., Nat Med 9, 129-134 (2003).

The term "antigen binding portion", "antigen binding domain" or "antigen binding portion of an antibody" as used herein refers to a portion of an antibody that includes a region that specifically binds to and is complementary to part or all of an antigen. The term therefore refers to the amino acid residues in an antibody that are responsible for antigen binding. The antigen binding domain can be provided by, for example, one or more antibody variable domains (also referred to as antibody variable regions). In particular, the antigen binding domain includes an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). The antigen binding portion of an antibody includes amino acid residues from a "complementarity determining region" or "CDR". The "framework" or "FR" region is those variable domain regions other than the hypervariable region residues defined herein. Therefore, the light chain variable domain and the heavy chain variable domain of an antibody include the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from N-terminus to C-terminus. In particular, the CDR3 of the heavy chain is the region that is most conducive to antigen binding and defines the characteristics of the antibody. CDR and FR regions are determined according to the standard definitions of Kabat et al. (Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991)) and/or those residues from the "hypervariable loop". The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, e.g., Kindt et al., Kuby Immunology, 6th Edition, W.H. Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity.

The term "epitope" refers to a protein determinant of an antigen that is capable of specific binding to an antibody, such as TYRP1 or human CD3. Epitopes are usually composed of chemically active surface macrogroups of molecules such as amino acids or sugar side chains, and usually epitopes have specific three-dimensional structural characteristics as well as specific charge characteristics. The difference between conformational and non-conformational epitopes is that in the presence of denaturing solvents, binding to the former but not the latter is lost.

The term "Fc domain" or "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. Although the boundaries of the IgG heavy chain Fc region may be slightly different, the human IgG heavy chain Fc region is generally defined as extending from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) in the Fc region may be present or absent. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, which is also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. The Fc domain of an antibody is not directly involved in the binding of the antibody to the antigen, but exhibits a variety of effector functions. "Fc domain of an antibody" is a term well known to the skilled person and is defined based on the cleavage of an antibody by papain. Antibodies or immunoglobulins can be classified into the following categories according to the different amino acid sequences of the constant regions of their heavy chains: IgA, IgD, IgE, IgG and IgM, and some of these antibodies can be further divided into subclasses (isotypes), such as IgG ₁ , IgG ₂ , IgG ₃ and IgG ₄ , IgA ₁ and IgA _2. Different classes of immunoglobulins are called α, δ, ε, γ and μ, respectively, according to the heavy chain constant region. The Fc domain of an antibody is directly involved in ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity) based on complement activation, C1q binding and Fc receptor binding. Complement activation (CDC) is initiated by the binding of complement factor C1q to the Fc domain of most IgG antibody subclasses. Although the effect of an antibody on the complement system depends on certain conditions, the binding to C1q is caused by a defined binding site in the Fc domain. Such binding sites are known in the prior art and are described, for example, by Boackle, RJ et al., Nature 282 (1979) 742-743; Lukas, T. J. et al., J. Immunol. 127 (1981) 2555-2560; Brunhouse, R. and Cebra, J. J., Mol. Immunol. 16 (1979) 907-917; Burton, DR et al., Nature 288 (1980) 338-344; Thommesen, J. E. et al., Mol. Immunol. 37 (2000) 995-1004; Idusogie, EE et al., J. Immunol. 164 (2000) 4178-4184; Hezareh, M. et al., J. Virology 75 (2001) 12161-12168; Morgan, A. et al., Immunology 86 (1995) 319-324; EP 0 307 434. Such binding sites are, for example, L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering according to the EU index of Kabat, EA, supra). Antibodies of the IgG ₁ , IgG ₂ and IgG ₃ subclasses generally exhibit complement activation and C1q and C3 binding, whereas IgG ₄ does not activate the complement system and does not bind C1q and C3.

In one embodiment, the antibody described herein comprises an Fc domain derived from human origin and all other parts of preferably human constant regions. As used herein, the term "Fc domain derived from human origin" means such an Fc domain, which is an Fc domain of a human antibody of IgG ₁ , IgG ₂ , IgG ₃ or IgG ₄ subclasses, preferably an Fc domain from a human IgG ₁ subclass, a mutant Fc domain from a human IgG 1 subclass, an Fc domain from a human IgG ₄ subclass or a mutant Fc domain from a human IgG ₄ subclass. In one embodiment, anti-TRYP1/anti-CD3 bispecific antibodies have reduced effector functions or minimal effector functions. In one embodiment, minimal effector functions are caused by Fc mutations without effectors. In one embodiment, Fc mutations without effectors are L234A/L235A or L234A/L235A/P329G or N297A or D265A/N297A. In one embodiment, the effector-free Fc mutations of each antibody are independently selected from the group consisting of L234A/L235A, L234A/L235A/P329G, N297A and D265A/N297A (EU numbering). In one embodiment, the TYRP1-specific antibody has ADCC activity in the presence of human effector cells, or has increased ADCC activity in the presence of human effector cells, compared to the otherwise identical antibody comprising a human wild-type IgG ₁ Fc region. In one embodiment, an antibody variant comprising an Fc region is provided, wherein the carbohydrate structure attached to the Fc region has reduced fucose or lacks fucose.

"Activating Fc receptors" are Fc receptors that, upon engagement of the Fc region of an antibody, cause signaling events that stimulate cells bearing the receptor to perform effector functions. Activating Fc receptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), and FcαRI (CD89). When used with respect to antibodies, the term "effector function" refers to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

In one embodiment, the antibodies described herein are of the human IgG class (ie, of the IgG ₁ , IgG ₂ , IgG ₃ or IgG ₄ subclass).

In preferred embodiments, the antibodies described herein are of human IgG ₁ subclass or human IgG ₄ subclass. In one embodiment, the antibodies described herein are of human IgG ₁ subclass. In one embodiment, the antibodies described herein are of human IgG ₄ subclass.

In one embodiment, the antibodies described herein are characterized in that the constant chain is of human origin. Such constant chains are well known in the prior art, for example, as described by Kabat, E.A. (see, for example, Johnson, G. and Wu, T.T., Nucleic Acids Res. 28 (2000) 214-218).

As used herein, the terms "first", "second" or "third" with respect to antibodies, Fc domain subunits, antigen binding portions, etc., are used to facilitate distinction when more than one different form of each type (i.e., different antibodies, Fc domain subunits, antigen binding portions) exists for each type. Unless explicitly stated, the use of these terms is not intended to confer a particular order or orientation.

As used herein, the term "nucleic acid" or "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA.

The term "amino acid" as used in this application refers to the group of naturally occurring carboxy α-amino acids including: alanine (three letter code: ala, single letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

"Percentage (%) of amino acid sequence identity relative to a reference polypeptide sequence is defined as after comparing a candidate sequence with a reference polypeptide sequence and introducing a vacancy (if necessary) to achieve the maximum percentage of sequence identity, and without considering any conservative substitution as a component of sequence identity, the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence. The comparison for determining amino acid sequence identity percentage can be achieved in various ways within the technical scope of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment on the full length of the compared sequence. However, for the purposes of this article, the sequence comparison computer program ALIGN-2 is used to generate amino acid sequence identity%. The ALIGN-2 sequence comparison computer program is written by Genentech, Inc., and the source code has been submitted to the U.S. Copyright Office, Washington D.C., 20559, with user documentation, where it is registered with U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged. In the case of amino acid sequence comparison using ALIGN-2, the amino acid sequence identity % of a given amino acid sequence A to a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or comprising a certain amino acid sequence identity % with a given amino acid sequence B) is calculated as follows:

Multiply 100 by the fraction X/Y

Where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the program's alignment of A and B, and where Y is the total number of amino acid residues in B. It should be understood that in the case where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless otherwise specifically indicated, all % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the previous paragraph. With respect to nucleic acids or polynucleotides having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention, it is meant that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, in order to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted by another nucleotide, or up to 5% of the number of nucleotides of the total nucleotides in the reference sequence may be inserted into the reference sequence. These changes to the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, or may be interspersed individually among the residues of the reference sequence, or may be interspersed in one or more consecutive groups within the reference sequence. As a practical matter, known computer programs, such as those discussed above for polypeptides (e.g., ALIGN-2), may be used to routinely determine whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotide sequence of the invention.

Methods of producing the antibodies described herein are provided, wherein the method comprises culturing a host cell comprising a polynucleotide encoding an antibody as provided herein under conditions suitable for expression of the antibody, and recovering the antibody from the host cell (or host cell culture medium).

The antibody at least comprises an antibody variable region capable of binding to an antigenic determinant. The variable region can form a part of and be derived from a naturally or non-naturally occurring antibody and its fragment. The method for producing polyclonal antibodies and monoclonal antibodies is well known in the art (see, for example, Harlow and Lane, "Antibodies, a laboratory manual", Cold Spring Harbor Laboratory, 1988). Non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be recombinantly produced (e.g., as described in U.S. Patent No. 4,186,567), or can be obtained, for example, by screening a combinatorial library comprising a variable heavy chain and a variable light chain (see, for example, U.S. Patent No. 5,969,108 of McCafferty). Antigen binding moieties and methods for producing them are also described in detail in PCT Publication WO 2011/020783, the entire contents of which are incorporated herein by reference.

Antibodies, antibody fragments, antigen-binding domains or variable regions of any animal species can be used in the antibodies described herein. Non-limiting antibodies, antibody fragments, antigen-binding domains or variable regions that can be used in the present invention can be of mouse, primate or human origin. If the antibody is intended for human use, a chimeric form of the antibody can be used, wherein the constant region of the antibody is from a human. Humanized or fully humanized antibodies can also be prepared according to methods well known in the art (see, e.g., U.S. Patent No. 5,565,332). Humanization can be achieved by various methods, including but not limited to (a) transplanting non-human (e.g., donor antibody) CDRs to human (e.g., acceptor antibody) frameworks and constant regions, which frameworks and constant regions have or do not retain key framework residues (e.g., key framework residues that are important for maintaining good antigen binding affinity or antibody function), (b) only transplanting non-human specific determining regions (SDR or a-CDR; residues that are critical to antibody-antigen interactions) to human frameworks and constant regions, or (c) transplanting entire non-human variable domains, but "hiding" them with human-like segments by replacing surface residues. Humanized antibodies and methods for their preparation are reviewed, e.g., in Almagro and Fransson, Front Biosci 13, 1619-1633 (2008), and further described, e.g., in Riechmann et al., Nature 332, 323-329 (1988); Queen et al., Proc Natl Acad Sci USA 86, 10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Jones et al., Nature 321, 522-525 (1986); Morrison et al., Proc Natl Acad Sci 81, 6851-6855 (1984); Morrison and Oi, Adv Immunol 44, 65-92 (1988); Verhoeyen et al., Science 239, 1534-1536 (1988); Padlan, Molec Immun 31(3), 169-217 (1994); Kashmiri et al., Methods 36, 25-34 (2005) (describing SDR(a-CDR) grafting); Padlan, Mol Immunol 28, 489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36, 43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36, 61-68 (2005) and Klimka et al., Br J Cancer 83, 252-260 (2000) (describing the "guided selection" method for FR shuffling). Various techniques known in the art can be used to produce human antibodies and human variable regions. Human antibodies are generally described in van Dijk and van de Winkel, Curr Opin Pharmacol 5, 368-74 (2001) and Lonberg, Curr Opin Immunol 20, 450-459 (2008). Human variable regions can form a part of a human monoclonal antibody prepared by a hybridoma method and be derived from the antibody (see, e.g., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)). Human antibodies and human variable regions can also be prepared in the following manner: an immunogen is administered to a transgenic animal that has been modified to produce a complete human antibody or complete antibody with a human variable region that responds to antigenic stimulation (see, e.g., Lonberg, Nat Biotech 23, 1117-1125 (2005)). Human antibodies and human variable regions can also be produced by isolating Fv clone variable region sequences selected from phage display libraries of human origin (see, e.g., Hoogenboom et al. Methods in Molecular Biology 178, 1-37 (O'Brien et al., eds., Human Press, Totowa, NJ, 2001); and McCafferty et al., Nature 348, 552-554; Clackson et al., Nature 352, 624-628 (1991)). Phage typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments.

In certain embodiments, antibodies are engineered to have enhanced binding affinity according to methods disclosed in, for example, PCT Publication WO 2011/020783 (see Examples related to affinity maturation) or US Patent Application Publication No. 2004/0132066, the entire contents of which are hereby incorporated by reference. The ability of an antibody to bind to a specific antigenic determinant can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (analyzed on a BIACORE T100 system) (Liljeblad, et al., Glyco J 17, 323-329 (2000)) and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). Competition assays can be used to identify antibodies, antibody fragments, antigen-binding domains, or variable domains that compete with a reference antibody for binding to a specific antigen. In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) as the reference antibody. Detailed exemplary methods for mapping epitopes bound by antibodies are provided in Morris (1996) "Epitope Mapping in Methods in Molecular Biology Vol. 66 (Humana Press, Totowa, NJ). In an exemplary competition assay, an immobilized antigen is incubated in a solution containing a first labeled antibody that binds to the antigen and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to the antigen. The second antibody may be present in the hybridoma supernatant. As a control, the immobilized antigen is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permissive for binding of the first antibody to the antigen, excess unbound antibody is removed and the amount of label associated with the immobilized antigen is measured. If the amount of label associated with the immobilized antigen in the test sample is significantly reduced relative to the control sample, it indicates that the second antibody is competing with the first antibody for binding to the antigen. See Harlow and Lane (1988) Antibodies: A Laboratory Manual Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

The anti-TYRP1/anti-CD3 bispecific antibodies described herein can be prepared as described in the examples of WO 2020/127619A1.

Antibodies as described herein are preferably produced by recombinant means. Such methods are well known in the prior art and include expressing proteins in prokaryotic and eukaryotic cells, subsequently isolating antibody polypeptides, and generally purifying them to pharmaceutical purity. For protein expression, nucleic acids encoding light and heavy chains or fragments thereof are inserted into expression vectors by standard methods. Expression is performed in appropriate prokaryotic or eukaryotic host cells such as CHO cells, NSO cells, SP2/0 cells, HEK293 cells, COS cells, yeast or Escherichia coli cells, and antibodies are recovered from cells (from supernatant or after cell lysis).

The recombinant production of antibodies is well known in the art and is reviewed, for example, in the following papers: Makrides, S.C., Protein Expr. Purif. 17 (1999) 183-202; Geisse, S. et al., Protein Expr. Purif. 8 (1996) 271-282; Kaufman, R.J., Mol. Biotechnol 16 (2000) 151-161; Werner, R.G., Drug Res. 48 (1998) 870-880.

The antibody may be present in whole cells, cell lysates, or in partially purified or substantially pure form. Purification is performed to eliminate other cellular components or other contaminants (e.g., other cellular nucleic acids or proteins) by standard techniques, including alkali/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art. See Ausubel, Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987), et al.

Expression in NS0 cells is described, for example, in Barnes, L.M. et al., Cytotechnology 32 (2000) 109-123; Barnes, L.M. et al., Biotech. Bioeng. 73 (2001) 261-270. Transient expression is described, for example, in Durocher, Y. et al., Nucl. Acids. Res. 30 (2002) E9. The cloning of variable domains is described, for example, in the following literature: Orlandi, R. et al., Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837; Carter, P., et al., Proc. Natl. Acad. Sci. USA 89 (1992) 4285-4289; Norderhaug, L., et al., J. Immunol. Methods 204 (1997) 77-87. A preferred transient expression system (HEK 293) is described, for example, in the following literature: Schlaeger, E.-J. and Christensen, K., Cytotechnology 30 (1999) 71-83; and Schlaeger, E.-J., J. Immunol. Methods 194 (1996) 191-199.

The heavy chain and light chain variable domains according to the present invention are combined with sequences of promoter, translation initiation, constant region, 3' untranslated region, polyadenylation and transcription termination to form an expression vector construct. The heavy chain and light chain expression constructs can be combined into a single vector, co-transfected, continuously transfected or transfected into a host cell separately and then fused to form a single host cell expressing the two chains.

Suitable control sequences for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, enhancers, and polyadenylation signals.

When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "operably linked". For example, the DNA of a presequence or secretory leader is operably linked to the DNA for a polypeptide (if it is expressed as a preprotein that participates in the secretion of the polypeptide); a promoter or enhancer is operably linked to a coding sequence that affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence that is positioned to facilitate translation. Generally, "operably linked" means that the DNA sequences connected are continuous and continuous and in the reading frame for the secretory leader sequence. However, enhancers do not have to be continuous. The connection is completed by connecting at a convenient restriction site. If such a site does not exist, a synthetic oligonucleotide adapter or linker is used according to conventional practice.

Monoclonal antibodies are suitable for separation from culture medium by conventional immunoglobulin purification methods (such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography). DNA and RNA encoding monoclonal antibodies are easy to be separated and sequenced by conventional methods. Hybridoma cells can be used as the source of such DNA and RNA. Once separated, DNA can be inserted into an expression vector, which is then transfected into a host cell such as HEK293 cell, CHO cell or myeloma cell that does not produce immunoglobulin in addition, to obtain the synthesis of recombinant monoclonal antibodies in the host cell.

As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably, and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard to the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content due to intentional or unintentional mutations. Variant progeny having the same function or biological activity as screened in the original transformed cell are included.

Methods of treatment and compositions

The present invention comprises a method for treating a patient in need of treatment, characterized by administering to the patient a therapeutically effective amount of a combination therapy of an anti-TYRP1/anti-CD3 bispecific antibody and a TYRP1-specific antibody.

The present invention comprises the use of the anti-TYRP1/anti-CD3 bispecific antibody and the TYRP1-specific antibody according to the present invention for the combination therapy.

A preferred embodiment of the present invention is the combination therapy of the anti-TYRP1/anti-CD3 bispecific antibody and the TYRP1-specific antibody of the present invention for treating cancer or tumors. Another embodiment of the present invention is the anti-TYRP1/anti-CD3 bispecific antibody described herein in combination with the TYRP1-specific antibody as described herein for treating tumors or cancer. Another embodiment of the present invention is the TYRP1-specific antibody described herein in combination with the anti-TYRP1/anti-CD3 bispecific antibody as described herein for treating cancer or tumors.

The term "cancer" as used herein can be, for example, lung cancer, non-small cell lung (NSCL) cancer, bronchoalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, In some embodiments, the present invention relates to cancer of the ovary, colon, breast, uterine, fallopian tube, endometrial, cervical, vaginal, vulvar, Hodgkin's disease, esophageal, small intestinal, endocrine system, thyroid, parathyroid, adrenal, soft tissue sarcoma, urethral, penile, prostate, bladder, kidney or ureteral, renal cell, renal pelvic, mesothelioma, hepatocellular, bile duct, central nervous system (CNS) tumors, spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, lymphoma, lymphocytic leukemia, including refractory forms of any of the above cancers, or a combination of one or more of the above cancers. In a preferred embodiment, such cancer is breast cancer, colorectal cancer, melanoma, head and neck cancer, lung cancer, or prostate cancer. In a preferred embodiment, such cancer is breast cancer, ovarian cancer, cervical cancer, lung cancer or prostate cancer. In another preferred embodiment, such cancer is breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma cancer, bladder cancer, kidney cancer, renal cancer, liver cancer, head and neck cancer, colorectal cancer, pancreatic cancer, gastric cancer, esophageal cancer, mesothelioma, prostate cancer, leukemia, lymphoma, myeloma. In a preferred embodiment, such cancer is a cancer expressing TYRP1.

An embodiment of the present invention is an anti-TYRP1/anti-CD3 bispecific antibody as described herein in combination with TYRP1 as described herein for treating any of the above cancers or tumors. Another embodiment of the present invention is an anti-TYRP1/anti-CD3 bispecific antibody as described herein in combination with TYRP1 as described herein for treating any of the above cancers or tumors. The present invention comprises a combination therapy of an anti-TYRP1/anti-CD3 bispecific antibody as described herein with a TYRP1-specific antibody as described herein for treating cancer. The present invention comprises a combination therapy of an anti-TYRP1/anti-CD3 bispecific antibody as described herein with a TYRP1-specific antibody as described herein for preventing or metastasis.

The present invention comprises a method for treating cancer in a patient in need thereof, characterized in that an anti-TYRP1/anti-CD3 bispecific antibody as described herein and a TYRP1-specific antibody as described herein are administered to the patient. The present invention comprises a method for preventing or treating metastasis in a patient in need thereof, characterized in that an anti-TYRP1/anti-CD3 bispecific antibody as described herein and a TYRP1-specific antibody as described herein are administered to the patient.

The present invention comprises an anti-TYRP1/anti-CD3 bispecific antibody as described herein in combination with a TYRP1 specific antibody as described herein for use in treating cancer, or alternatively for the manufacture of a medicament for treating cancer in combination with a TYRP1 specific antibody as described herein.

The present invention comprises an anti-TYRP1/anti-CD3 bispecific antibody as described herein in combination with a TYRP1 specific antibody as described herein for use in preventing or treating metastasis, or alternatively for the manufacture of a medicament for preventing or treating metastasis in combination with a TYRP1 specific antibody as described herein.

The present invention comprises a TYRP1 specific antibody as described herein in combination with an anti-TYRP1/anti-CD3 bispecific antibody as described herein for use in treating cancer, or alternatively for the manufacture of a medicament for treating cancer in combination with an anti-TYRP1/anti-CD3 bispecific antibody as described herein.

The present invention comprises a TYRP1 specific antibody as described herein in combination with an anti-TYRP1/anti-CD3 bispecific antibody as described herein for use in preventing or treating metastasis, or alternatively for the manufacture of a medicament for preventing or treating metastasis in combination with an anti-TYRP1/anti-CD3 bispecific antibody as described herein.

In a preferred embodiment of the present invention, the anti-TYRP1/anti-CD3 bispecific antibody for combined treatment and medical use of the above-mentioned different diseases is an anti-TYRP1/anti-CD3 bispecific antibody characterized by comprising the polypeptide sequences of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8, and the TYRP1-specific antibody used in such combined treatment is characterized by comprising the polypeptide sequences of SEQ ID NO:13 and SEQ ID NO:14.

In another aspect, the present invention provides a composition, such as a pharmaceutical composition, comprising an anti-TYRP1/anti-CD3 bispecific antibody described herein and a TYRP1-specific antibody described herein formulated together with a pharmaceutically acceptable carrier.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption/resorption delaying agents, etc. that are physiologically compatible. Preferably, the carrier is suitable for injection or infusion.

The compositions of the present invention can be administered by a variety of methods known in the art. As will be appreciated by those skilled in the art, the route and/or mode of administration will vary depending on the desired results.

Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for preparing sterile injectable solutions or dispersions. Such media and agents are known in the art for use in pharmaceutically active substances. In addition to water, the carrier can also be, for example, an isotonic buffered saline solution.

Regardless of the administration route selected, the compound of the present invention and/or the pharmaceutical composition of the present invention, which can be used in a suitable hydrated form, can be formulated into a pharmaceutical dosage form by conventional methods known to those skilled in the art.

The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration without causing toxicity to the patient (effective amount). The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the present invention employed, or its ester, salt or amide, the route of administration, the time of administration, the excretion rate of the particular compound employed, other drugs, compounds and/or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical field.

In one aspect, the present invention provides a kit intended for the treatment of a disease, comprising (a) an anti-TYRP1/anti-CD3 bispecific antibody as described herein, and (b) a TYRP1-specific antibody as described herein, in the same container or separate containers, and optionally further comprising (c) a package insert including printed instructions for using the combination therapy as a method for treating the disease. In addition, the kit may include (a) a first container containing a composition, wherein the composition comprises an anti-TYRP1/anti-CD3 bispecific antibody as described herein; (b) a second container containing a composition, wherein the composition comprises a TYRP1-specific antibody as described herein; and optionally (c) a third container containing a composition, wherein the composition comprises an additional cytotoxic agent or other therapeutic agent. The kit in this embodiment of the present invention may also include a package insert indicating that the composition can be used to treat a specific condition. Alternatively or in addition thereto, the kit may further include a third (or fourth) container containing a pharmaceutical buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The kit may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

In one aspect, the present invention provides a kit for treating a disease, comprising (a) a container comprising an anti-TYRP1/anti-CD3 bispecific antibody as described herein, and (b) a package insert comprising instructions directing the use of the anti-TYRP1/anti-CD3 bispecific antibody in combination therapy with a TYRP1-specific antibody as described herein as a method of treating the disease.

In another aspect, the present invention provides a kit for treating a disease, comprising (a) a container comprising a TYRP1-specific antibody as described herein, and (b) a package insert comprising instructions directing the use of the TYRP1-specific antibody in combination therapy with an anti-TYRP1/anti-CD3 antibody as described herein as a method of treating the disease.

In a further aspect, the present invention provides a medicament for treating a disease comprising an anti-TYRP1/anti-CD3 antibody as described herein, wherein the medicament is for use in combination therapy with a TYRP1-specific antibody as described herein, and optionally comprises a package insert comprising printed instructions directing the use of the combination therapy as a method of treating the disease.

In yet a further aspect, the invention provides a medicament for treating a disease comprising a TYRP1-specific antibody as described herein, wherein the medicament is for use in a combination therapy with an anti-TYRP1/anti-CD3 antibody as described herein, and optionally comprises a package insert comprising printed instructions directing the use of the combination therapy as a method of treating the disease

The term "treatment" or its equivalent, when applied to, for example, cancer, refers to a procedure or course of action intended to reduce or eliminate the number of cancer cells in a patient, or to alleviate the symptoms of cancer. A "treatment" of cancer or another proliferative disease does not necessarily mean that the cancer cells or other disease will actually be eliminated, that the number of cells or the disease will actually be reduced, or that the cancer or other disease will actually be put into remission. In general, a treatment for cancer may have a low probability of success but is still considered to induce an overall beneficial course of action, given the patient's medical history and estimated survival expectancy.

The terms "combined administration" or "co-administration", "co-administration", "combination therapy" or "combination treatment" refer to the administration of an anti-TYRP1 / anti-CD3 bispecific antibody as described herein and a TYRP1-specific antibody as described herein, for example, as separate formulations / applications (or as a single formulation / application). Co-administration can be performed simultaneously or sequentially in any order, wherein preferably there is a time period during which both (or all) active agents exert their biological activities simultaneously. The active agents are co-administered simultaneously or sequentially by continuous infusion (e.g., intravenously (i.v.)). When two therapeutic agents are co-administered sequentially, they can be administered in two separate administrations on the same day, or one of the agents can be administered on day 1 and the second agent can be co-administered on day 2 to day 7, preferably day 2 to day 4. Therefore, in one embodiment, the term "sequentially" means within 7 days after administration of the first component, preferably 4 days after administration of the first component; and the term "simultaneously" means preferably at the same time. The term "co-administered" with respect to maintenance doses of anti-TYRP1/anti-CD3 antibody and/or TYRP1-specific antibody means that if the treatment cycle is suitable for both drugs, for example, weekly, the maintenance doses can be co-administered simultaneously. Alternatively, the maintenance doses are co-administered sequentially, for example, doses of anti-TYRP1/anti-CD3 antibody and TYRP1-specific antibody are given every other week.

It is understood that the antibody is administered to a patient in a "therapeutically effective amount" (or simply "effective amount"), which is the amount of the corresponding compound or combination that will elicit the biological or medical response of a tissue, system, animal or human that the researcher, veterinarian, medical doctor or other clinician is seeking.

The amount of co-administration and the timing of co-administration will depend on the type (species, sex, age, weight, etc.) and condition of the patient being treated and the severity of the disease or condition being treated. The anti-TYRP1/anti-CD3 antibody and/or TYRP1-specific antibody is suitably co-administered to the patient at one time or over a series of treatments, e.g., on the same day or one day after or once a week.

In addition to the anti-TYRP1/anti-CD3 antibodies in combination with the TYRP1-specific antibodies, chemotherapeutic agents may also be administered.

In one embodiment, such additional chemotherapeutic agents that can be administered with the anti-TYRP1/anti-CD3 antibodies as described herein and the TYRP1-specific antibodies as described herein include, but are not limited to, anti-tumor agents including alkylating agents, including: nitrogen mustards such as dichloromethyl diethylamine, cyclophosphamide, ifosfamide, melphalan and chlorambucil; nitrosoureas such as carmustine (BCNU), lomustine (CCNU) and semustine (methyl CCNU); Temodal ^™ (temozolomide), ethyleneimine/methylmelamines such as triethylene melamine (TEM), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, hexamethylmelamine); alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC); antimetabolites including folic acid analogs such as methotrexate and trimetrexate, pyrimidine analogs such as 5-fluorouracil (5FU), fluorodeoxyuridine, gemcitabine, cytarabine (AraC, cytarabine), 5-azacytidine, 2,2'-difluorodeoxycytidine, purine analogs such as 6-mercaptopurine, 6-thioguanine, azathioprine, T-deoxycoformycin (pentostatin), erythrohydroxynonyl adenine (EHNA), fludarabine phosphate and 2-chlorodeoxyadenosine (cladribine, 2-CdA); natural products, including antimitotic drugs such as paclitaxel, vinca alkaloids (including vinblastine (VLB) , vincristine and vinorelbine), taxotere, estramustine and estramustine phosphate; podophyllotoxins such as etoposide and teniposide; antibiotics such as actinomycin D, daunomycin (erythromycin), doxorubicin, mitoxantrone, idarubicin, bleomycin, plicamycin (mithramycin), mitomycin C and actinomycin; enzymes such as L-asparaginase; biological response modifiers such as interferon-α, IL-2, G-CSF and GM-CSF; Miscellaneous drugs, including platinum coordination complexes (such as oxaliplatin, cisplatin and carboplatin), anthracenediones (such as mitoxantrone), substituted ureas (such as hydroxyurea), methylhydrazine derivatives (including N-methylhydrazine (MIH) and procarbazine), adrenocortical suppressants (such as mitotane (o,p-DDD) and aminoglutethimide); hormones and antagonists, including adrenocortical steroid antagonists, such as prednisone and its equivalents, dexamethasone and aminoglutethimide; Gemzar ^™ (gemcitabine), progestins, such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogens, such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogens, such as tamoxifen; androgens, including testosterone propionate and fluoxymesterone/equivalents; antiandrogens, such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; and nonsteroidal antiandrogens, such as flutamide. Therapies targeting epigenetic mechanisms include, but are not limited to, histone deacetylase inhibitors, demethylating agents (eg, Vidaza), and transcriptional inhibition release (ATRA) therapy, and may also be combined with antigen binding proteins. In one embodiment, the chemotherapeutic agent is selected from the group consisting of taxanes (e.g., paclitaxel (Taxol), docetaxel (Taxotere), modified paclitaxels (e.g., Abraxane and Opaxio), doxorubicin, sunitinib (Sutent), sorafenib (Nexavar) and other multikinase inhibitors, oxaliplatin, cisplatin and carboplatin, etoposide, gemcitabine and vinblastine. In one embodiment, the chemotherapeutic agent is selected from the group consisting of taxanes (e.g., paclitaxel, docetaxel (Taxotere), modified paclitaxels (e.g., Abraxane and Opaxio). In one embodiment, the additional chemotherapeutic agent is selected from 5-fluorouracil (5-FU), folinic acid, irinotecan or oxaliplatin. In one embodiment, the chemotherapeutic agent is 5-fluorouracil, folinic acid and irinotecan (FOLFIRI). In one embodiment, the chemotherapeutic agent is 5-fluorouracil and oxaliplatin (FOLFOX).

Specific examples of combination therapies with additional chemotherapeutic agents include, e.g., therapies for the treatment of breast cancer: taxanes (e.g., docetaxel or paclitaxel) or modified paclitaxels (e.g., Abraxane or Opaxio, doxorubicin), capecitabine and/or bevacizumab (Avastin); therapies for ovarian cancer using carboplatin, oxaliplatin, cisplatin, paclitaxel, doxorubicin (or modified doxorubicin (Caelyx or Doxil)), or topotecan (Hycamtin); therapies for the treatment of renal cancer using multikinase inhibitors, MKIs (Sutent, Nexavar, or 706), and/or doxorubicin; therapies for the treatment of squamous cell carcinoma using oxaliplatin, cisplatin, and/or radiation; therapies for the treatment of lung cancer using paclitaxel and/or carboplatin.

Thus, in one embodiment, the additional chemotherapeutic agent is selected from the group consisting of taxanes (docetaxel or paclitaxel or modified paclitaxel (Abraxane or Opaxio), doxorubicin, capecitabine and/or bevacizumab for the treatment of breast cancer.

In one embodiment, the anti-TYRP1/anti-CD3 antibody and TYRP1-specific antibody combination therapy is one in which a chemotherapeutic agent is not administered.

The present invention also includes methods for treating a patient suffering from such a disease as described herein.

The present invention also provides a method for manufacturing a pharmaceutical composition comprising an effective amount of an anti-TYRP1/anti-CD3 antibody according to the present invention as described herein and a TYRP1-specific antibody according to the present invention as described herein and a pharmaceutically acceptable carrier, as well as the use of the anti-TYRP1/anti-CD3 antibody and TYRP1-specific antibody according to the present invention as described herein for such a method.

The present invention also provides the use of an effective amount of an anti-TYRP1/anti-CD3 antibody according to the invention as described herein and a TYRP1-specific antibody according to the invention as described herein for the manufacture of a pharmaceutical medicament for treating a patient suffering from cancer, preferably together with a pharmaceutically acceptable carrier.

Cell therapy

In some embodiments, the immunotherapy is activated immunotherapy. In some embodiments, the immunotherapy is provided as a cancer treatment. In some embodiments, the immunotherapy comprises adoptive cell transfer.

In some embodiments, adoptive cell transfer includes administering T cells expressing chimeric antigen receptors (CART cells). Technicians will understand that CAR is an antigen targeting receptor consisting of an intracellular T cell signaling domain fused to an extracellular tumor binding portion, most commonly a single-chain variable fragment (scFv) from a monoclonal antibody.

CAR directly recognizes cell surface antigens, independent of MHC-mediated presentation, allowing the use of a single receptor construct for any given antigen in all patients. The original CAR fused the antigen recognition domain to the CD3 activation chain of the T cell receptor (TCR) complex. Although these first-generation CARs induce T cell effector functions in vitro, they are largely limited by poor anti-tumor efficacy in vivo. Subsequent CAR iterations include secondary co-stimulatory signals in series with CD3, including intracellular domains from CD28 or various TNF receptor family molecules, such as 4-1BB (CD137) and OX40 (CD134). In addition, the third-generation receptor includes two co-stimulatory signals in addition to CD3, the most common of which are CD28 and 4-1BB. The second and third generation CARs significantly improve the anti-tumor efficacy, inducing complete remission in patients with advanced cancer in some cases. In one embodiment, a CAR T cell is an immune response cell modified to express a CAR, which is activated when the CAR binds to its antigen.

In one embodiment, a CAR T cell is an immune response cell comprising an antigen receptor, which is activated when its receptor binds to its antigen. In one embodiment, the CAR T cell used in the compositions and methods disclosed herein is a first generation CAR T cell. In another embodiment, the CAR T cell used in the compositions and methods disclosed herein is a second generation CAR T cell. In another embodiment, the CAR T cell used in the compositions and methods disclosed herein is a third generation CAR T cell. In another embodiment, the CAR T cell used in the compositions and methods disclosed herein is a fourth generation CART cell.

In some embodiments, adoptive cell transfer includes administering T cell receptor (TCR) modified T cells. The skilled person will understand that TCR modified T cells are made by isolating T cells from tumor tissue and isolating their TCRa and TCRβ chains. These TCRa and TCRβ are then cloned and transfected into T cells isolated from peripheral blood, and then the T cells express TCRa and TCRβ from T cells that recognize the tumor.

In some embodiments, adoptive cell transfer includes administration of tumor infiltrating lymphocytes (TIL). In some embodiments, adoptive cell transfer includes administration of chimeric antigen receptor (CAR) modified NK cells. Technicians will understand that CAR modified NK cells include NK cells isolated from patients or commercially available NK cells engineered to express CARs that recognize tumor-specific proteins.

In some embodiments, adoptive cell transfer comprises administering dendritic cells.

In some embodiments, immunotherapy comprises administering a cancer vaccine. As will be appreciated by the skilled artisan, a cancer vaccine exposes the immune system to cancer-specific antigens and adjuvants. In some embodiments, the cancer vaccine is selected from the group comprising: sipuleucel-T, GVAX, ADXS11-001, ADXS31-001, ADXS31-164, ALVAC-CEA vaccine, AC vaccine, talimogene laherparepvec, BiovaxID, Prostvac, CDX110, CDX1307, CDX1401, CimaVax-EGF, CV9104, DNDN, NeuVax, Ae-37, GRNVAC, tarmogens, GI-4000, GI-6207, GI-6301, ImPACT therapy, IMA901, hepcortespenlisimut-L, Stimuvax, DCVax-L, DCVax-Direct, DCVax prostate, CBLI, Cvac, RGSH4K, SCIB1, NCT01758328 and PVX-410.

The following examples, sequence listings and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It should be understood that modifications may be made to the procedures set forth without departing from the spirit of the present invention.

Further examples:

A further embodiment of the present invention is a novel TYRP1-specific antibody comprising a heavy chain variable domain VH of SEQ ID NO:1 and a light chain variable domain VL of SEQ ID NO:2.

In one embodiment, the TYRP1-specific antibody is of human _IgG1 subclass.

In another embodiment, the TYRP1-specific antibody comprises a polypeptide sequence of SEQ ID NO:13 and a polypeptide sequence of SEQ ID NO:14; or a polypeptide sequence of SEQ ID NO:15 and a polypeptide sequence of SEQ ID NO:16.

Examples

Production and purification of muTYRP1 IgG

Since human _IgG1 molecules induce immunogenic responses in mice, glycosylated muIgG2a was used as a surrogate molecule for animal experiments.

The heavy and light chains of the antibody were cloned into a plasmid controlled by a human CMV promoter-intron A-5'UTR box. The BGH polyadenylation signal is located downstream of the gene. The rat GnTIII gene was amplified from a rat kidney cDNA library (BD Biosciences) by polymerase chain reaction (PCR). The gene encoding ManII was amplified from human DNA using gene-specific primers. Both genes were combined with a chimeric MPSV promoter and subcloned into an expression vector. The vector was derived from pUC18 (Thermo Fisher Scientific) and expression was enhanced by inserting a puromycin resistance gene or a hygromycin resistance gene and a scaffold attachment region (SAR).

CHO-K1SV cells were transfected with plasmids encoding anti-TYRP1 muIgG2a heavy and light chains and N-acetylglucosaminyltransferase-III (GntIII) and mannosidase-II (ManII) enzymes. Stable clones were selected and fed-batch cultured in serum-free medium for up to 14 days to produce antibodies with modified glycosylation structures. Cell supernatants were harvested by centrifugation and subsequent filtration (0.2 μm filter), and antibodies were purified from the harvested supernatants using standard methods as shown below.

Proteins were purified from filtered cell culture supernatants according to standard protocols. Briefly, Fc-containing proteins were purified from cell culture supernatants using protein A affinity chromatography (equilibrium buffer: 20 mM sodium citrate, 20 mM sodium phosphate, pH 7.5; elution buffer: 20 mM sodium citrate, 100 mM NaCl, 100 mM glycine, 0.01% Tween 20, pH 3.0). Elution was achieved at pH 3.0, followed by immediate neutralization of the sample pH. The elution was performed by centrifugation (Millipore Proteins were concentrated using ULTRA-15 (Art. Nr.: UFC903096) and aggregated proteins were separated from monomeric proteins using size exclusion chromatography in 20 mM histidine, 140 mM sodium chloride, 0.01% Tween 20, pH 6.0.

The concentration of purified protein was determined by measuring the absorbance at 280 nm, using the mass extinction coefficient calculated based on the amino acid sequence according to the method described in Pace et al. (Protein Science, 1995, 4, 2411-1423). In the presence and absence of a reducing agent, the purity and molecular weight of the protein were analyzed by CE-SDS using LabChip GXII or LabChip GX Touch (Perkin Elmer) (Perkin Elmer). Aggregation content was determined by HPLC chromatography at 25 ° C using an analytical size exclusion column (TSKgel G3000 SW XL or UP-SW3000) equilibrated in running buffer (200 mM KH2PO4, 250 mM KCl pH 6.2, 0.02% NaN3). To determine the level of α-fucosylation, N-linked oligosaccharides were cleaved from purified IgG by incubation with 0.005 U PNGase F (QAbio, USA) and EndoH (QAbio, USA) in 20 mM Tris pH 8.0 for 16 hours at 37° C. This produced free oligosaccharides, which were analyzed by MALDI TOF mass spectrometry (Autoflex, Bruker Daltonics) in positive ion mode according to Papac et al. (1996) Anal. Chem., 68: 3215-3223.

Table 1: Monomeric product peak, high molecular weight (HMW) and low molecular weight (LMW) by-products determined by analytical size exclusion chromatography.

Table 1: Major product peaks determined by non-reducing CE-SDS.

Table 2: Carbohydrate α-fucosylation levels determined by MALDI-TOF MS analysis.

Construct TAPIR ID a-fuc(%) TYRP1 TA99 muIgG2a glycosylation modification P1AA9559-007 52

Glycosylated TYRP1 mouse IgG2a was purified by protein A and size exclusion chromatography. Quality analysis of the purified material showed that the monomer content was 99% by analytical size exclusion chromatography (Table 1), the main product peak was 95% by non-reduced capillary electrophoresis (Table 2), and the α-fucosylation level was 52% by MALDI-TOF MS analysis (Table 3). Therefore, glycosylated TYRP1 mouse IgG2a can be produced with high quality, and the α-fucosylation level is sufficient to ensure increased ADCC.

In vivo efficacy of anti-TYRP1/anti-CD3 bispecific antibodies alone and in combination with TYRP1-IgG antibodies in a mouse tumor cell line syngeneic model.

The anti-tumor efficacy of anti-TYRP1/anti-CD3 bispecific antibody (TYRP1-TCB) in combination with TYRP1-IgG antibody was tested in the B16-muFAP-Fluc metastatic melanoma syngeneic model. The murine surrogates muTYRP1-TCB (SEQ ID NOs: 9, 10, 11 and 12) and muTYRP1-IgG (SEQ ID NOs: 15 and 16) were tested in Black 6 albino mice injected intravenously with the mouse melanoma B16-muFAP-Fluc dual metastatic cell line.

B16 cells (mouse melanoma cells) were initially obtained from ATCC (Manassas, VA, USA) and stored in the Roche-Glycart internal cell bank after amplification. The B16-muFAP-Fluc cell line was produced internally by calcium transfection and subcloning technology. B16-muFAP-Fluc was cultured in RPMI medium containing 10% FCS (Sigma), 200 μg/ml zeocin, 0.75 μg/ml hygromycin and 1% Glutamax. Under 5% CO ₂ , cells were cultured at 37°C in a water-saturated atmosphere. The 13th generation was used for transplantation. Cell viability was 94.3%. 2x 10 ⁵ cells per animal were injected intravenously (iv) using a 0.3ml tuberculin syringe (BD Biosciences, Germany). Two hundred microliters of cell suspension (2x10 ⁵ B16-muFAP-Fluc cells in RPMI medium) were injected into the tail vein.

Female Black 6 albino mice (Charles Rivers, Lyon, France), 8–10 weeks old at the start of the experiment, were maintained under specific pathogen-free conditions with a 12 h light/12 h dark daily cycle according to regulatory guidelines (GV-Solas; Felasa; TierschG). The experimental study protocol was reviewed and approved by the local authorities (ZH225/2017). After arrival, the animals were maintained for one week to acclimate to the new environment and were observed. Continuous health status monitoring was performed regularly.

Mice were injected intravenously with 2 x 10 ⁵ B16-muFAP-Fluc cells on study day 0, randomized and weighed. Eighteen days after tumor cell injection, mice were injected intravenously once a week for 4 weeks with muTYRP1-TCB or muTYRP1-IgG single agents and compared with the combination of muTYRP1-TCB + muTYRP1-IgG.

All mice were injected intravenously with 200 μl of the appropriate solution. Mice in the vehicle group were injected with histidine buffer, and the treatment groups were injected with 10 mg/kg of muTYRP1-TCB and 20 mg/kg of muTYRP1-IgG. To obtain the appropriate amount of immunoconjugate per 200 μl, the stock solution was diluted with histidine buffer when necessary.

Table 4: Compounds used in the examples

Figure 1 shows that the combination of muTYRP1-TCB and muTYRP1-IgG had significantly superior efficacy in enhancing median and overall survival compared to all other treatment groups and the vehicle group.

sequence

Claims (18)

1. A combination of an anti-TYRP 1/anti-CD 3 bispecific antibody with a second TYRP 1-specific antibody for use in a combination therapy for the treatment of cancer, for use in a combination therapy for the prevention or treatment of metastasis,

Wherein the anti-TYRP 1/anti-CD 3 bispecific antibody comprises a first antigen-binding portion comprising a heavy chain variable domain VH of SEQ ID NO:1 and a light chain variable domain VL of SEQ ID NO:2 specifically binding to TYRP1 and a second antigen-binding portion comprising a heavy chain variable domain VH of SEQ ID NO:3 and a light chain variable domain VL of SEQ ID NO:4 specifically binding to CD3, and

Wherein the second TYRP 1-specific antibody comprises an antigen-binding portion that specifically binds to TYRP 1.

2. The use of a combination of an anti-TYRP 1/anti-CD 3 bispecific antibody with a second TYRP1 specific antibody in the manufacture of a medicament for the treatment of cancer,

Wherein the anti-TYRP 1/anti-CD 3 bispecific antibody comprises a first antigen-binding portion comprising a heavy chain variable domain VH of SEQ ID NO:1 and a light chain variable domain VL of SEQ ID NO:2 specifically binding to TYRP1 and a second antigen-binding portion comprising a heavy chain variable domain VH of SEQ ID NO:3 and a light chain variable domain VL of SEQ ID NO:4 specifically binding to CD3, and

Wherein the second TYRP 1-specific antibody comprises an antigen-binding portion that specifically binds to TYRP 1.

3. A method of treating cancer in an individual, the method comprising administering to the individual a therapeutically effective amount of a combination of an anti-TYRP 1/anti-CD 3 bispecific antibody and a second TYRP 1-specific antibody,

Wherein the anti-TYRP 1/anti-CD 3 bispecific antibody comprises a first antigen-binding portion comprising a heavy chain variable domain VH of SEQ ID NO:1 and a light chain variable domain VL of SEQ ID NO:2 specifically binding to TYRP1 and a second antigen-binding portion comprising a heavy chain variable domain VH of SEQ ID NO:3 and a light chain variable domain VL of SEQ ID NO:4 specifically binding to CD3, and

Wherein the second TYRP 1-specific antibody comprises an antigen-binding portion that specifically binds to TYRP 1.

4. The combination of an anti-TYRP 1/anti-CD 3 bispecific antibody for use according to claim 1 with a second TYRP 1-specific antibody, the use according to claim 2 or the method according to claim 3, wherein the second antibody comprises the heavy chain variable domain VH of SEQ ID No. 1 and the light chain variable domain VL of SEQ ID No. 2.

5. The combination, use or method of an anti-TYRP 1/anti-CD 3 bispecific antibody for use according to any one of the preceding claims with a second TYRP 1-specific antibody, wherein the anti-TYRP 1/anti-CD 3 bispecific antibody is a human IgG ₁ or a human IgG ₄ subclass.

6. The combination, use or method of an anti-TYRP 1/anti-CD 3 bispecific antibody for use according to any one of the preceding claims with a second TYRP 1-specific antibody, wherein the TYRP 1-specific antibody is a human IgG ₁ subclass.

7. The combination, use or method of an anti-TYRP 1/anti-CD 3 bispecific antibody for use according to any one of the preceding claims with a second TYRP 1-specific antibody, wherein the anti-TYRP 1/anti-CD 3 bispecific antibody has reduced or minimal effector function.

8. The combination, use or method of an anti-TYRP 1/anti-CD 3 bispecific antibody for use according to any one of the preceding claims with a second TYRP 1-specific antibody, wherein the minimal effector function is caused by an Fc mutation of a null effector.

9. The combination, use or method of an anti-TYRP 1/anti-CD 3 bispecific antibody for use according to claim 8 with a second TYRP 1-specific antibody, wherein the effector-free Fc mutation is L234A/L235A or L234A/L235A/P329G or N297A or D265A/N297A.

10. The combination, use or method of an anti-TYRP 1/anti-CD 3 bispecific antibody for use according to any one of the preceding claims with a second TYRP1 specific antibody, characterized in that the second TYRP1 specific antibody comprises an Fc domain with improved effector function, in particular improved ADCC function.

11. The combination, use or method of an anti-TYRP 1/anti-CD 3 bispecific antibody for use according to any one of the preceding claims with a second TYRP 1-specific antibody, wherein the second TYRP 1-specific antibody is defucosylated.

12. The combination, use or method of an anti-TYRP 1/anti-CD 3 bispecific antibody for use according to any one of the preceding claims with a second TYRP 1-specific antibody, wherein the anti-TYRP 1/anti-CD 3 bispecific antibody comprises:

i) A polypeptide sequence of SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID No. 7 or SEQ ID No. 8,

Ii) the polypeptide sequences of SEQ ID NO.5 and SEQ ID NO. 6 and SEQ ID NO. 7 and SEQ ID NO. 8, or

Iii) Polypeptide sequences of SEQ ID NO. 9 and SEQ ID NO. 10 and SEQ ID NO. 11 and SEQ ID NO. 12.

13. The combination, use or method of an anti-TYRP 1/anti-CD 3 bispecific antibody for use according to any one of the preceding claims with a second TYRP 1-specific antibody, wherein the anti-TYRP 1/anti-CD 3 bispecific antibody comprises:

i) A polypeptide sequence of SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID No. 7 or SEQ ID No. 8,

Ii) the polypeptide sequences of SEQ ID NO.5 and SEQ ID NO. 6 and SEQ ID NO. 7 and SEQ ID NO. 8, or

Iii) Polypeptide sequences of SEQ ID NO. 9 and SEQ ID NO. 10 and SEQ ID NO. 11 and SEQ ID NO. 12,

And wherein the second TYRP 1-specific antibody comprises:

i) The polypeptide sequence of SEQ ID NO. 13 or SEQ ID NO. 14, or

Ii) the polypeptide sequences of SEQ ID NO. 13 and SEQ ID NO. 14; or (b)

Iii) The polypeptide sequences of SEQ ID NO. 15 and SEQ ID NO. 16.

14. A combination of an anti-TYRP 1/anti-CD 3 bispecific antibody and a second TYRP 1-specific antibody for use in:

i) Inhibiting tumor growth in a tumor; and/or

Ii) increasing the median and/or overall survival of a subject suffering from a tumor;

Wherein the anti-TYRP 1/anti-CD 3 bispecific antibody comprises:

i) A polypeptide sequence of SEQ ID No. 5 or SEQ ID No. 6 or SEQ ID No. 7 or SEQ ID No. 8,

Ii) the polypeptide sequences of SEQ ID NO.5 and SEQ ID NO. 6 and SEQ ID NO. 7 and SEQ ID NO. 8, or

Iii) Polypeptide sequences of SEQ ID NO. 9 and SEQ ID NO. 10 and SEQ ID NO. 11 and SEQ ID NO. 12,

And wherein the second TYRP 1-specific antibody comprises:

i) The polypeptide sequence of SEQ ID NO. 13 or SEQ ID NO. 14, or

Ii) the polypeptide sequences of SEQ ID NO. 13 and SEQ ID NO. 14; or (b)

Iii) The polypeptide sequences of SEQ ID NO. 15 and SEQ ID NO. 16.

15. The combination, use or method of an anti-TYRP 1/anti-CD 3 bispecific antibody for use according to any one of the preceding claims with a second TYRP 1-specific antibody, wherein the cancer is selected from the group of: breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma cancer, bladder cancer, kidney cancer, liver cancer, head and neck cancer, colorectal cancer, melanoma, pancreatic cancer, gastric cancer, esophageal cancer, mesothelioma, prostate cancer, leukemia, lymphoma, myeloma.

16. The combination, use or method of an anti-TYRP 1/anti-CD 3 bispecific antibody for use according to any one of the preceding claims with a second TYRP 1-specific antibody, wherein the patient is treated or pre-treated with immunotherapy.

17. The combination, use or method of an anti-TYRP 1/anti-CD 3 bispecific antibody with a second TYRP 1-specific antibody for use according to claim 16, wherein the immunotherapy comprises adoptive cell transfer, administration of monoclonal antibodies, administration of cytokines, administration of cancer vaccines, T cell engagement therapy, or any combination thereof.

18. The combination, use or method of an anti-TYRP 1/anti-CD 3 bispecific antibody with a second TYRP 1-specific antibody for use according to claim 17, wherein the adoptive cell transfer comprises administering a T cell expressing a chimeric antigen receptor (CAR T cell), a T Cell Receptor (TCR) modified T cell, a Tumor Infiltrating Lymphocyte (TIL), a Chimeric Antigen Receptor (CAR) modified natural killer cell, a T Cell Receptor (TCR) transduced cell, or a dendritic cell, or any combination thereof.